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* Instituto de Investigaciones Bioquímicas, Universidad Nacional del Sur-CONICET, Bahía Blanca, Argentina; and Departamento de Física, Universidad Nacional del Sur, Bahía Blanca, Argentina
Correspondence: Address reprint requests to Dr. Cecilia Bouzat, Instituto de Investigaciones Bioquímicas, Camino La Carrindanga, Km 7-B8000FWB, Bahía Blanca, Argentina. Fax: 54-291-4861200; E-mail: inbouzat{at}criba.edu.ar.
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ABSTRACT |
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2ß
), each having four transmembrane domains (M1–M4). The atomic model of the nicotinic acetylcholine receptor shows that the pore-lining M2 domains make no extensive contacts with the rest of the transmembrane domains. However, there are several sites where close appositions between segments occur. It has been suggested that the pair M1-F15' and M2-L11' is one of the potential interactions between segments. To determine experimentally if these residues are interacting and to explore if this interhelical interaction is essential for channel gating, we combined mutagenesis with single-channel kinetic analysis. Mutations in M1-F15' lead to profound changes in the opening rate and slighter changes in the closing rate. Channel gating is impaired as the volume of the residue increases. Rate-equilibrium linear free-energy relationship analysis reveals an 
70% open-state-like environment for M1-F15' at the transition state of the gating reaction, suggesting that it moves early during the gating process. Replacing the residue at M1-15' by that at M2-11' and vice versa profoundly alters gating, but the combination of the two mutations restores gating to near normal, indicating that M1-F15' and M2-L11' are interchangeable. Double-mutant cycle analysis shows that these residues are energetically coupled. Thus, the interaction between M1 and M2 plays a key role in channel gating. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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). The AChR is a pentamer of homologous subunits with a composition 2ß in the adult muscle. Each subunit is composed of an extracellular domain that contains the binding sites and four transmembrane (TM) segments (M1–M4). Electron microscopy images of AChR at 4-Å resolution revealed that the four TM domains have -helical structures (2). The M2 segment of each subunit delineates the ion channel pore, which contains the gate that allows the pore to switch from ion impermeable to ion permeable (2). M1, M3, and M4 are located behind M2, forming an external shield that isolates the pore from the lipid membrane.
The binding of the agonist triggers a concerted, global change in the protein's conformation that results in the opening of the channel gate. The mechanism of channel gating is still not clear. The interface between the extracellular and TM domains is essential to couple agonist binding to channel gating (2,3–8). Channel gating primarily involves motion of the M2 segments, which leads to the broadening of the ion pore (2). The motions of M1, M3, and M4 segments during channel gating are less understood. However, several experimental data support their contribution to gating kinetics (9–16). Rate-equilibrium free-energy relationship analyses (REFER) have provided evidence about the structural dynamics of TM segments during channel gating (17,18). These studies have suggested that M2 moves asynchronously, with the rearrangement of the extracellular half preceding that of the middle part during opening (4,19,20). In contrast, M4 moves as a synchronous unit, near the middle of the gating reaction (16). Residues at M2 and M4 of ß, , and subunits move later during the gating reaction than the equivalent residues of subunits (16,20,21).
We have recently shown that M1 contributes to gating and that mutations at position 15' of M1 of the ß subunit lead to significant changes in kinetics (13). Kinetic analysis of ßM1-I15'F AChR channels activated by choline revealed a 28-fold increase in the gating equilibrium constant of the diliganded receptor and a significant increased opening in the absence of agonist. REFER analysis suggested an 70% closed-state like environment for the ß15' position at the transition state of gating.
The atomic model of the closed pore of the AChR shows that although M2 makes no extensive van der Waals contacts with the other TM segments, there are several sites where close appositions between segments occur. It has been suggested that the pair M1-F15' and M2-L11' is one of the potential interactions between segments (2). Interactions between residues located at different regions of the AChR are essential for the dynamics of channel gating (5,7,22–24). Thus, identifying pairwise interactions between TM domains that contribute to channel gating will allow us to better understand the gating process.
Here we study in detail the structural contribution of position 15' of M1 of the , , and subunits of the muscle AChR to channel gating. In addition, we explore the interaction of this position with 11' of M2, and we describe how this interaction affects channel gating.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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, ß, , and AChR subunits were used (25). Mutant subunits were constructed using the Quik Change site-directed mutagenesis kit (Stratagene, La Jolla, CA). Restriction mapping and DNA sequencing confirmed all constructs. The L269F mutation, associated with a slow-channel congenital myasthenic syndrome (26), was used as a background mutation in some experiments.
Expression of AChR
BOSC 23 cells (24) were transfected with , ß, , and cDNA subunits (wild-type or mutant) using calcium phosphate precipitation at a subunit ratio of 2:1:1:1 for /ß//, respectively, essentially as described previously (25,27). For transfections, cells at 40–50% confluence were incubated for 8–12 h at 37°C with the calcium phosphate precipitate containing the cDNAs in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum. Cells were used for single-channel measurements 1 or 2 days after transfection.
Patch-clamp recordings
Recordings were obtained in the cell attached configuration (28) at a membrane potential of –70 mV and at 20°C. The bath and pipette solutions contained 142 mM KCl, 5.4 mM NaCl, 1.8 mM CaCl2, 1.7 mM MgCl2, and 10 mM HEPES, pH 7.4. Patch pipettes were pulled from 7052 capillary tubes (Garner Glass, Claremont, CA) and coated with Sylgard (Dow Corning, Midland, MI). Acetylcholine (ACh) or choline was added to the pipette solution.
Single-channel currents were recorded using an Axopatch 200B patch-clamp amplifier (Axon Instruments, Union City, CA), digitized at 5-µs intervals with the PCI-6111E interface (National Instruments, Austin, TX), recorded to the hard disk of a computer using the program Acquire (Bruxton Corporation, Seattle, WA), and detected by the half-amplitude threshold criterion using the program TAC (Bruxton Corporation) at a final bandwidth of 10 kHz. Open- and closed-time histograms were plotted using a logarithmic abscissa and a square root ordinate and fitted to the sum of exponential functions by maximum likelihood using the program TACFit (Bruxton Corporation). Open probability within clusters (Popen) was determined experimentally by calculating the mean fraction of time that the channel is open within a cluster.
Data of AChRs activated by 20 mM choline were analyzed at a bandwidth of 5 kHz to avoid detection of blockages that could be resolved at 10 kHz. Owing to open-channel block, AChRs activated by 20 mM choline show a 60% reduction in channel amplitude (29). At –70 mV, single-channel amplitudes of wild-type receptors activated by 30 µM ACh and 20 mM choline were 5.50 ± 0.14 and 2.10 ± 0.04 pA, respectively. Current amplitudes determined at 100 µM choline were similar to those determined at 30 µM ACh. The KB for fast blockade by choline, calculated according to io/ib=1+[B]/KB where io is the current at low agonist concentration, ib is the current at 20 mM choline, [B] is choline concentration (20 mM), and KB is the dissociation equilibrium constant for binding to the blocking site and is 12 mM, in good agreement with recent reports (30).
Kinetic analysis
Kinetic analysis was performed as described before (9,10,14). The analysis was restricted to clusters of channel openings, each reflecting the activity of a single AChR. Clusters were identified as a series of closely spaced events preceded and followed by closed intervals longer than a critical duration (
crit), which was taken as the point of intersection of the predominant closed component and the succeeding one in the closed-time histogram. Similar results were obtained when crit was calculated by solving numerically for crit in the expression exp(–crit/2) = [1 –exp(–crit/3)] by using MAPLE 7 (Waterloo Maple, Ontario, Canada), where 2 is the predominant longest closed component within clusters and 3 is the succeeding one (31,32).
Only clusters containing more than 10 openings were considered for further analysis. In addition, clusters showing double openings were rejected. For mutations that decrease the Popen, particularly M2-L11', recordings with extremely low channel activity were used to allow a better identification of clusters. To this end, the cells were incubated with the calcium phosphate precipitate overnight, and recordings were started at different times immediately after changing the medium until channel activity appeared.
The resulting open and closed intervals from single patches at 20 mM choline were analyzed according to kinetic schemes using an interval-based full likelihood algorithm (www.qub.buffalo.edu; QuB suite, State University of New York, Buffalo, NY). Briefly, the program allows simultaneous fitting of recordings and estimates the rate constants using a maximum likelihood method that corrects for missed events (33). Calculated rates were accepted only if the resulting probability density functions correctly fitted the experimental open- and closed-duration histograms.
For the analysis, we fitted dwell times from the selected clusters by the kinetic scheme containing one open and one closed state given that 20 mM choline is a saturating concentration (29,34). Increasing choline concentration up to 20 mM increases 2-fold the apparent mean open time compared with that obtained at low concentrations (11,20,30). Because the single-channel amplitude was reduced to a similar extent in wild-type and all mutant AChRs, we assumed that the mutations do not change fast blockade for choline and therefore the prolongation of the openings is the same for all constructs.
Double-mutant cycle analysis
Gating equilibrium constants (
2) obtained from kinetic analysis was used to calculate the coupling coefficient 
based on Eq. 1:
 | (1) |
):
 | (2) |
Rate-equilibrium free-energy relationships
We applied REFER analysis to determine the structure of the gating transition state near position 15' of the subunit (16,17,29,36). For this analysis, the values calculated for rate (ß2) and gating equilibrium (2) constants were used. The correlation between the rate and equilibrium constants for series of point mutants, 
, measures the extent to which the perturbed region at the reaction transition state resembles the open conformation. is a fraction between 1 and 0, with = 1 implying an open-like character.
Molecular modeling
The refined coordinates of Torpedo AChR at 4-Å resolution (Protein Data Bank (PDB) with accession code 2BG9) (37) were used as a starting model to evaluate the structural implications of the mutations. Single and double point mutations of the amino acids located at 15' of M1 and 11' of M2 were modeled with the program O (38), and the geometry regularization of the resulting structures was performed with REFMAC (39). The geometry of the final models was examined with the program PROCHECK (40). Figures were prepared with Visual Molecular Dynamics (41).
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RESULTS |
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). We here explore the structural contribution of the equivalent position in , , and subunits to channel gating. To this end, we replaced the residues by amino acids with different side chains and evaluated kinetic changes.
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). All the mutations in the subunit, except F15'Y, increase the mean open time (Table 1). Mutations in the and subunits lead to both increased and decreased mean open times (Table 1). At 30 µM ACh, the closed time distributions show a major intermediate component of 1.5 ms, whose duration is dependent on ACh concentration and corresponds to closings within clusters (Fig. 2) (9). The duration of this main closed component decreases from 1.8- (I) to 145-fold (W) in the F15' mutant receptors with respect to wild-type (Table 1). The probability of channel opening (Popen) increases in all M1-15' mutant AChRs, except in the F15'Y (Table 1). In contrast, mutations at 15' of the and subunits slightly change (F) significantly decrease (A) or increase (Y) the Popen (Table 1).
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). Because the Popen increases in the M1 mutants, we used choline as an agonist to estimate the kinetics of activation for the different AChRs. Choline is a weak agonist of the muscle AChR, which shows a diliganded gating equilibrium constant that is 500 times smaller than ACh (29,34).
Activation by choline
Channel activity of wild-type and mutant AChRs activated by 20 mM choline occurs in clusters (Fig. 3). At this choline concentration, channel amplitude decreases 60% (11,29). Open- and closed-time histograms show a single component (Fig. 3 and Table 1). Mean open times at 20 mM choline are highly variable among M1-15' mutants, showing a 12-fold difference between the briefest and the longest durations (Fig. 3 and Table 1). Mean closed times decrease in all M1 mutants, except in F15'Y, and show a 500-fold difference between the briefest and longest durations (Table 1). As described for ACh, the Popen within clusters increases in all M1-15' mutants, except in the F15'Y (Table 1).
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). To analyze the kinetics of channel opening and closing, the closed and open intervals of the selected clusters were fitted by Scheme 1 (Fig. 3) (10,21):
 | (Scheme 1) |
The curves resulting from the kinetic analysis superimposed on the experimental open and closed duration histograms (Fig. 3). The analysis reveals a great increase in the opening rate, ß2, when M1-F15' is mutated (Table 1). The exception is tyrosine, which decreases this rate. The gating equilibrium constant, 2, covers a wide range of values, which show a 1900-fold difference between the lowest and the highest one (Table 1). In the and subunits, the replacement of I15' by Y increases the gating equilibrium constant whereas alanine leads to an impairment of channel gating (Table 1).
Structural contribution of position M1-15' to channel gating
To determine if there is a correlation between the chemical properties of the residue at 15' of M1 and the changes in channel gating, we plotted 2 against the volume and the hydrophobicity of the residue (Fig. 4). Regarding the subunit, with the exception of W, a strong correlation between 2 and the amino acid volume is observed. Similarly, the gating equilibrium constant seems to correlate with the volume of the amino acid at 15' of the and subunits. It is interesting to note that inverse relationships are observed between and non- subunits; i.e., 2 decreases as a function of the volume in the subunit whereas it increases in non- subunits.
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M1 mutant AChRsM1 (16,36). For the analysis we used the values of the opening rate and gating equilibrium constant shown in Table 1. The BrØnsted plot obtained using ß2 versus 2 for substitutions is shown in Fig. 5. The slope of the linear relationship, , estimates the extension to which the mutated site has adopted its open structure at the transition state of the gating reaction (17). We obtained a value of 0.74 ± 0.06 (r2=0.97) for mutations in 15' of M1, indicating that the structure of the transition state at this position resembles the open state in 70%. Zhou et al. (17) have recently derived the analytical form of a REFER for a linear chain of coupled reactions and showed that the experimental REFERs appear to be more linear than those predicted by the theory. The reason for this discrepancy is still unknown. We therefore corroborated that the data can be well fitted by a second order polynomial: y = yo + ax + bx2 (r2 = 0.99), resulting in a = 0.68 ± 0.04 and b = –0.14 ± 0.04. The value for thus calculated (0.68) is similar to that calculated from a linear plot.
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M1-F15' is close to M2-L11' (2) (Fig. 1). To determine experimentally if these residues are interacting, we generated pairs of reverse mutations within subunits and recorded channels activated by 20 mM choline from the single- (M1-F15'L and M2-L11'F) and double-mutant receptors (M1-F15'L+M2-L11'F) (Fig. 6). Although position 11' of M2 does not face the ion channel pore, we first analyzed how the mutation at this position affects channel blockade induced by high choline concentrations. To this end, we determined single-channel amplitudes of wild-type and single- and double-mutant AChRs activated by low (100 µM) and high (20 mM) concentrations of choline. Current amplitudes for 100 µM and 20 mM choline at –70 mV were 5.50 ± 0.11 and 2.15 ± 0.04 pA (wild-type), 5.31 ± 0.10 and 2.12 ± 0.10 pA (M2-L11'F), and 5.27 ± 0.15 and 2.11 ± 0.07 pA (M1-F15'I+M2-L11'F). Therefore, we can ensure that in the M2 mutant the blockade by 20 mM choline is similar to that of wild-type AChR.
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17-fold and increases 10-fold in the M1-F15'L and M2-L11'F, respectively. In contrast, the mean closed time of the double mutant is similar to that of wild-type AChRs (Fig. 6 and Table 2). The kinetic analysis revealed that the opening rate increases 15-fold in the single M1 mutant and decreases 10-fold in the M2 mutant but it is similar to that of wild-type in the double mutant (Table 2). Thus, the mutation in M1 improves channel gating, whereas that in M2 impairs channel gating (Table 2). The combined mutations, which restore the pair of interacting residues, restore gating to near normal (Table 2). Thus, F15' in M1 and L11' in M2 are interchangeable. We calculated the changes due to the mutations in the free energy of the gating equilibrium of diliganded AChRs. Whereas M1-F15'L decreases the free energy (–1.9 kcal/mol), the mutation M2-L11'F increases it (1.4 kcal/mol). The change in the free energy of the double mutant (0.7 kcal/mol) is significantly different from the sum of the changes occurring in the two single mutants. This result indicates that the effects of the mutations are not independent and that the residues are coupled in their contribution to gating (35). To test further for interaction between M1-F15' and M2-L11' and to quantify energetic coupling between them we analyzed the changes in the free energy of coupling by double-mutant thermodynamic cycles (Fig. 7). When the gating equilibrium constants, 2, are cast as a mutant cycle, the free energy of coupling is –1.2 kcal/mol. The analysis confirms that the residues at 15' of M1 and 11' of M2 interact with each other.
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M1-F15'L AChRs activated by ACh is too fast to be measured reliably. However, due to the low Popen of M2-L11'F activated by choline, recordings in which clusters could be well distinguished were difficult to obtain (see Materials and Methods). In addition, the determination of the low opening rate constant of this mutant may yield inaccurate values. Therefore, to corroborate the value obtained for ß2, we first combined the M2 mutation with another one that increases the opening rate. We chose the L269F mutation, a gain of function mutation which has been associated to a slow-channel congenital myasthenic syndrome (26). As shown in Fig. 8 and Table 2, L269F-containing channels activated by 20 mM choline show longer openings and briefer closings, and clusters of channel openings can be clearly distinguished. The decrease in channel amplitude due to choline block is similar to that of wild-type AChRs (2.10 ± 0.08 pA), indicating that channel block is not affected by the mutation L269F. In all AChRs containing the background mutation, the closed-time histograms constructed with the selected clusters show a second closed component, which is clearly noticeable in the double mutant (Fig. 8). We do not know the origin of this closed component, but for other mutants it has been proposed that a second closed component may originate from a short-lived desensitized state (20). We analyzed the kinetics of channel opening and closing on the basis of Scheme 2, which is an extension of Scheme 1 and includes a second closed state:
 | (Scheme 2) |
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L269F mutation increases ß2 and decreases 2 (Table 2). When the M2-L11'F mutation is introduced into this template receptor, clusters can be well distinguished (Fig. 8). The kinetic analysis shows that the mutation decreases the opening rate of the template receptor 8-fold (Table 2). Such decrease is similar to that observed in the M2-L11'F with respect to the wild-type receptor (9.5-fold). We also compared the decrease in ß2 due to the M2-L11'F mutation in channels activated by ACh. The data obtained at a range of 100–1000 µM ACh were well fitted by the classical kinetic scheme for ACh activation (9). The kinetic analysis showed that ß2 decreased from its wild-type value of 50,000 s–1 (9,42) to 7000 ± 300 s–1, which corresponds to a 7.1-fold change. Therefore, we can ensure that despite the low Popen of M2-L11'F, the estimate for ß2 shown in Table 2 does not contain a high degree of uncertainty.
The fast opening rate of the L269F+M1-F15'L double mutant did not allow the kinetic analysis. Nevertheless, we determined that the changes in 2, ß2, and 2 for the receptor containing the double M1-F15'L+M2-L11'F mutation together with the L269F mutation are similar to those of the double mutant in the absence of the background mutation with respect to the corresponding template receptor (Table 2). The rates of closing and reopening from the second closed state (A2R' in Scheme 2) are similar for all receptors containing the L269F mutation. These rates were d+1: 180 ± 50, 160 ± 30, and 120 ± 50 s–1 for the L269F, the double mutant (L269F+M2-L11'F), and the triple mutant (L269F+M1-F15'L+M2-L11'F), respectively; d-1: 6800 ± 3400, 8900 ± 2500, and 3700 ± 200 s–1 for the L269F, the double mutant (L269F+M2-L11'F), and the triple mutant (L269F+M1-F15'L+M2-L11'F), respectively. The effects of the M2-L11'F mutation are not significantly affected by the background L269F mutation, as the change in the free energy of the gating equilibrium of the double mutant (L269F+M2-L11'F) is similar to the sum of the changes occurring in the two single mutants.
To obtain a structural view of how the mutations affect the interaction between positions 11' of M2 and 15' of M1, we modeled the structure of single- and double-mutant AChRs using the refined structure of the Torpedo AChR (PDB ID 2BG9) (37). As expected, when F15' in M1 is replaced by L, there is more free space in the cavity as the volume of the amino acid is reduced from 189.9 Å3 to 166.7 Å3. The replacement of M2-L11' by F yields several possible conformations. One of them results in an edge-to-face interaction between the mutant phenylalanine residue in M2 and M1-F15'. The model shows that the benzyl rings of both phenylalanine residues are separated by 3.6 Å (centroid-centroid distance) and form an edge-to-face interaction with an angle of 66° (Fig. 9). This type of interaction is commonly observed in proteins and it may have a substantial role in protein stabilization (43–46). An alternative orientation of the introduced phenylalanine residue forming a displaced face-to-face interaction with M1-F15' is also sterically possible. Finally, the modeling of the double-mutant M1-F15'L+M2-L11'F shows that the interface between M1 and M2 is not substantially affected when the two residues are exchanged (Fig. 9).
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DISCUSSION |
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subunits and isoleucine in non- subunits (Fig. 1). We have previously studied in detail the contribution to channel gating of this position in the ß subunit (13). Here we have extended the study to , , and subunits. We determined that in the subunit this position interacts with position 11' of the M2 domain, and we showed the importance of such interaction for appropriate channel gating.
Mutations at position 15' of M1 lead to receptors that show a wide spectrum of mean open, mean closed, and Popen values. Because the opening rate may increase in the mutants and because such increase cannot be accurately determined using ACh as an agonist, we used choline to determine channel kinetics (13,29,34). The changes in mean open time, mean closed time, and Popen follow a qualitatively similar but not quantitatively identical pattern in the mutants activated by either ACh or choline.
The diliganded gating equilibrium constant for choline, 2, increases in all M1 mutants, except in F15'Y. With respect to wild-type AChRs, such increase varies from 1.8-fold in the F15'I to 540-fold in the F15'W. The high ratio between the smallest and largest gating equilibrium constant values suggests that this position of M1 undergoes a pronounced change in its environment between the closed and open conformations. A similar behavior has been reported for residues at the external half of the M2 segment (21). The changes in 2 arise from a significant increase in the opening rate and a less profound decrease in the closing rate of the mutant with respect to wild-type AChRs (Table 1). There is a spatial gradient in the extent to which AChR mutations that change the gating equilibrium constant alter the opening versus the closing rate constant (20). More significant changes in the opening than in the closing rates, as shown here for F15', occur for mutations at positions that move earlier during channel gating. For example, the opening rate is significantly affected by mutations at residues located at the extracellular half of M2, whereas the closing rate is more affected by mutations at the equatorial position (position 9') (20).
Our results show a strong correlation between the volume of the residue at position 15' and its contribution to gating. For the subunit, except for tryptophan, channel gating is enhanced as a function of the decrease in the volume of the amino acid. Out of eight different residues, only tryptophan shows an anomalous behavior. Although this residue has a large side chain, its presence at position 15' dramatically enhances gating. One explanation for this behavior may be related to the unique properties of W. W prefers to have six partners around it with which it may form different types of interactions (46). Its indole ring can form aromatic 
interactions to other aromatic rings, to positively charged side-chains, and/or to adjacent C-H or N-H, whereas its indole amide can function as a hydrogen-bond donor (47). Thus, it is possible that more extensive arrangements may occur in the mutant structure to accommodate the W at 15'. In addition to the volume, there is also some influence of the stereochemistry of the side chain on channel gating. This is revealed from the differences in 2 between mutants containing L or I, which have the same volume, but L lacks the asymmetric ß carbon atom. A similar result, showing that both the volume and the stereochemistry of the side chain contribute to channel gating, has been reported for V285 in M3 (15).
Residues in the and subunits also contribute to gating. Interestingly, their contribution is correlated with the volume in an opposite way to that observed in the subunit: channel gating is enhanced by increasing the volume of the residue.
Diliganded AChR gating occurs as a reversible conformational wave that connects transmitter-binding sites and the gate (36). We used REFER analysis to estimate , a fraction between 0 and 1 that quantifies the extent to which the mutated region at the gating reaction transition state resembles the open conformation (20,36). In this context, values provide insight into the dynamics of AChR gating, suggesting the sequence of movement of residues during the gating process since the receptor binds the agonist (16). The temporal significance of the value of has been recently confirmed by simulation and theoretical studies (17,18). A value close to 1 indicates that the residue moves early during the gating process, whereas a value close to 0 indicates that the residue moves late during this process. The REFER is linear for position 15' of M1, with a slope corresponding to a value of 0.70. We have previously determined a value of 0.27 for position 15' of ßM1 (13), thus indicating that position 15' in the subunit moves earlier than the same position in ß.
It has been shown that residues appear to be organized into contiguous domains within which all members have similar values (18). These domains may or may not overlap secondary structural elements and may move synchronously as a unit or block (16,20). Interestingly, the value obtained for M1-15' is similar to that calculated for the upper half of M2 (0.65) (20), suggesting that these residues may move at the same time during gating. values are similar at the extracellular positions 17' and 27' of M2 and they are smaller at the equatorial 9' position (20). Although the value at 11' of M2 has not been determined, it might be similar to that of residues at the extracellular half. In agreement with this, mutations in the upper half of M2 affect more the opening than the closing rate, as shown here for M2-L11'F. Thus, the fact that M1-15' shows a value similar to that of the upper half of M2 agrees with our observation that the interaction between M1-15' and M2-11' is essential for appropriate channel gating, as these residues may move in block during such process.
M1 is located behind M2, forming an external ring together with M3 and M4. The structural model at 4-Å resolution (2) suggests that M2 makes minimal contacts with the other TM segments. One close apposition between M1 and M2 was reported to occur at M2-L253 (position 11') and M1-F225 (position 15') (2). The interaction between these residues may affect the relative movements of the inner and outer rings during gating. By exchanging residues between M1 and M2 segments and performing double-cycle mutant analysis, we experimentally determined that such interaction is taking place and that it plays a key role in channel gating. The kinetic analysis revealed that the single mutations at these residues affect mainly the opening rate and that this change occurs in opposite directions in the different segments. Thus, whereas the mutation in M1 improves channel opening, that in M2 impairs it. The kinetic changes are counteracted when both single mutations are combined. The double-mutant receptor, which contains the original pair of residues, shows normal gating kinetics. Thus, F15' in M1 and L11' in M2 are interchangeable in their contributions to gating. The change in free energy of gating for the double mutant differs from the sum of the changes of the two single mutants, indicating that the mutated residues are coupled (35). The double-mutant cycle analysis has been used to explore pairwise interactions between residues (48–50). When applied here, it revealed that these residues interact with each other with a coupling energy of –1.2 kcal/mol.
Modeling of the single and double mutants illustrated the experimental results. The structure of the AChR at 4-Å resolution shows that M2 is mainly separated from the other helices by water-filled spaces (2). The minimal contacts of M2 with the rest of the segments may favor the movement of M2 during gating. Thus, the increase in the volume of the side chain at the M1-M2 interacting site, as shown in the M2-L11'F mutant receptor as well as in the M1 mutants, might restrict such movements, thus resulting in impaired channel gating as described here. Accordingly, in the M1-F15'L mutant, the presence of two leucine residues at the site of interaction allows M2 to move more freely, favoring channel opening. Restoring the wild-type pair of residues in the double-mutant AChR, which shows a similar structure at the M1-M2 interface, leads to normal gating.
The L-F pair is the most abundant interhelical pairwise interaction in TM regions of membrane proteins (51). Here we show that in the AChR, this interaction is essential for appropriate channel gating probably by connecting the M2 movement to the M1 movement. Finally, understanding how the TM helices interact with each other will help us to understand how the assembled receptors carry out their biological functions.
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ACKNOWLEDGEMENTS |
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Submitted on May 10, 2006; accepted for publication September 15, 2006.
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), where 
represents the gating equilibrium constant for the single or double-mutant receptors. The free energy for coupling (
